Project Summary Cells migrating in tissues, including cancer cells, use filopodia to guide them through the 3D environment and increased formation of filopodia correlates strongly with the metastatic potential and invasiveness of cancer cells. Filopodia are slender actin-filled projections composed of a core of cross-linked, parallel actin bundles. They are highly dynamic, vary in length and found in a wide variety of cell types such as neurons that use them for gradient sensing and efficient directional migration or cancer cells that employ them for moving out from tumors into neighboring tissue. The first steps of filopodia formation are not well understood. Three conserved proteins are required for their formation - a MyTH4-FERM myosin (MF; MyTH4 = myosin tail homology 4; FERM = band 4.1, ezrin, radixin, moesin) and two regulators of actin polymerization, VASP and Formin. How the action of these three proteins is coordinated to initiate filopodia formation is unknown. The objective of this proposal is to define the molecular mechanism of filopodia initiation with an emphasis on the role of a MF myosin in this process. The versatile model system, Dictyostelium will be used to define how a MF myosin and VASP work together to organize the fast growing ends of actin filaments at the membrane to initiate polymerization. A combination of in vivo, in vitro and in silico approaches will be employed to a) determine the functional relationship between a MF myosin, VASP and formin, b) identify the specific properties of the myosin motor used for filopodia initiation, and c) develop a stochastic computational model with predictive power that will inform the experimental goals, the results of which will be used to refine the model. The knowledge generated by this project will reveal how cells use a myosin-based motor to build specific actin-based structures such as filopodia. Understanding how initiation occurs will also reveal how cells control filopodia formation to undergo directed migration or invade into surrounding tissues.